Promoters, expression cassettes, vectors, kits, and methods for the treatment of achromatopsia and other diseases

ABSTRACT

The present invention provides isolated promoters, transgene expression cassettes, vectors, kits, and methods for treatment of genetic diseases that affect the cone cells of the retina.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/269,723, filed on May 5, 2014, which claims priority to U.S.Provisional Application 61/824,071, filed on May 16, 2013. The entirecontents of each of the above documents is herein incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

Achromatopsia is an autosomal recessive retinal disease characterized bymarkedly reduced visual acuity, nystagmus, severe photophobia underdaylight conditions, and reduced or complete loss of colordiscrimination (Kohl, S. et al. Achromatopsia. In: Pagon R A, Bird T C,Dolan C R, Stephens K, editors Gene Reviews [Internet]. Seattle:University of Washington; 2010). It may be partial or complete. SeePang, J.-J. et al. (2010). Achromatopsia as a Potential Candidate forGene Therapy. In Advances in Experimental Medicine and Biology, Volume664, Part 6, 639-646 (2010) (hereinafter Pang et al). Symptoms ofachromatopsia include reduced visual acuity, achromatopia (lack of colorperception), hemeralopia (reduced visual capacity in bright lightaccompanied by photoaversion, meaning a dislike or avoidance of brightlight), nystagmus (uncontrolled oscillatory movement of the eyes), irisoperating abnormalities, and impaired stereovision (inability toperceive three-dimensional aspects of a scene). Electroretinogramsreveal that in achromatopsia, the function of retinal rod photoreceptorsremains intact, whereas retinal cone photoreceptors are not functional.Mutations in the cone-specific cyclic nucleotide gated channel betasubunit (CNGB3) gene account for about 50% of cases of achromatopsia(Kohl S, et al. Eur J Hum Genet 2005; 13:302-8). The rod and conephotoreceptors serve functionally different roles in vision. Pang et al.(2010). Cone photoreceptors are primarily responsible for central, fineresolution and color vision while operating in low to very bright light.They are concentrated in the central macula of the retina and comprisenearly 100% of the fovea. Rod photoreceptors are responsible forperipheral, low light, and night vision; they are found primarilyoutside the macula in the peripheral retina.

Approximately 1 in 30,000 individuals suffers from completeachromatopsia. In complete achromatopsia, there is total color visionloss, central vision loss, and visual acuity of 20/200 or worse. Thus,most individuals with achromatopsia are legally blind. The currentstandard of care consists of limiting retinal light exposure with tintedcontact lenses and providing magnification to boost central vision.However, there is no treatment available that corrects cone function inachromatopsia. Pang et al.

There are various genetic causes of congenital achromatopsia. Mutationsin the cyclic nucleotide-gated ion channel beta 3 (CNGB3, also known asACHM3) gene, are one genetic cause of achromatopsia. Recent studies indogs suggest some promise for the use of recombinant adeno-associatedvirus (rAAV)-based gene therapy for the treatment of achromatopsiacaused by mutations in the CNGB3 gene. Komaromy et al., Gene therapyrescues cone function in congenital achromatopsia. Human MolecularGenetics, 19(13): 2581-2593 (2010) (hereinafter Komaromy et al.). In thecanine studies, the rAAV vectors that were used packaged a human CNGB3(hCNGB3) expression cassette that contained elements including a 2.1 kbcone red opsin promoter (PR2.1) and a human CNGB3 (hCNGB3) cDNA. Onelimitation of the studies is that the hCNGB3 driven by the PR2.1promoter is expressed only in red and green cones, whereas endogenoushCNGB3 is expressed in all three types of cones (red, green and bluecones). Another limitation is that the overall size of the expressioncassette utilized (5,230 bp) was well beyond the normal packagingcapacity (<4.9 kb) of AAV particles; the over-stuffed rAAV particlesdramatically impaired the rAAV packaging efficiency, resulting in lowyields, a higher empty-to-full particle ratio, and likely a lowerinfectivity of the vector. Expression cassettes containing a shorterversion of the cone red opsin promoter, or a cone arrestin promoter,were much less effective in restoring visual function. The presentinvention addresses these limitations.

The present invention has the advantage of providing promoters that arecapable of promoting hCNGB3 expression in all three types of cones. Inaddition, the promoters of the invention have the advantage that theyare short enough to make the hCNGB3 expression cassette fit well withinthe normal packaging capacity of rAAV. A promoter that fits within thenormal rAAV packaging capacity provides benefits, such as improvedyields, a lower empty-to-full particle ratio, and higher infectivity ofthe vector. The present invention also provides expression cassettes,vectors and kits that utilize these improved promoters, as well asmethods for treating achromatopsia by administering the vectors.

The present invention addresses the need for an effective achromatopsiatreatment.

SUMMARY OF THE INVENTION

The present invention features, in a first aspect, a nucleic acidcomprising a portion of the cone cell specific promoter PR 2.1.

In one embodiment, the nucleic acid comprises the sequence SEQ ID NO: 4.In another embodiment, PR2.1 is truncated at the 5′ or the 3′ end. In arelated embodiment, the truncation is between about 100 base pairs to1,500 base pairs. In a further related embodiment, the truncation isabout 300 base pairs at the 5′ end. In another further embodiment, thetruncation is about 500 base pairs at the 5′ end. In another embodiment,the truncation is about 1,1000 base pairs at the 5′ end. In anotherfurther embodiment, the truncation is about 300 base pairs at the 3′end. In another embodiment, the truncation is about 500 base pairs atthe 3′ end. In another further embodiment, the truncation is about1,1000 base pairs at the 3′ end.

In one embodiment, the nucleic acid of the above aspects and embodimentscomprises SEQ ID NO:3. In one embodiment, the nucleic acid of the aboveaspects and embodiments comprises SEQ ID NO:2. In one embodiment, thenucleic acid of the

In another aspect, the invention features a nucleic acid comprising thenucleotide sequence of SEQ ID NO:1. In another aspect, the inventionfeatures a nucleic acid comprising the nucleotide sequence of SEQ IDNO:2. In another aspect, the invention features a nucleic acidcomprising the nucleotide sequence of SEQ ID NO:3.

In another further embodiment, the invention features a nucleic acidcomprising a nucleotide sequence which is at least 85% identical to thenucleotide sequence of SEQ ID NO:1.

In one embodiment of the above aspects, the promoter is capable ofpromoting CNGB3 expression in S-cone cells, M-cone cells, and L-conecells. In another embodiment of the above aspects, the promoter iscapable of promoting CNGA3 expression in S-cone cells, M-cone cells, andL-cone cells. In yet another embodiment of the above aspects, thepromoter is capable of promoting GNAT2 expression in S-cone cells,M-cone cells, and L-cone cells.

In another embodiment, the invention features a recombinantadeno-associated (rAAV) expression vector comprising a target nucleicacid sequence operably linked to the nucleic acid of any one of theabove aspects and embodiments. In a related embodiment, the rAAV isserotype 1. In a related embodiments, the rAAV is serotype 2. In anotherrelated embodiment, the rAAV is serotype 5. In still another relatedembodiment, the rAAV is comprised within an AAV virion.

In one embodiment, the target nucleic acid sequence encodes a cyclicnucleotide-gated channel subunit B (CNGB3) polypeptide. In a relatedembodiment, the CNGB3 is mouse CNGB3. In another related embodiment, theCNGB3 is rat CNGB3. In still another related embodiment, the CNGB3 ishuman CNGB3.

In one embodiment, the target nucleic acid sequence encodes a cyclicnucleotide-gated channel subunit A (CNGA3) polypeptide. In a relatedembodiment, the CNGA3 is mouse CNGA3. In another related embodiment, theCNGA3 is rat CNGA3. In still another related embodiment, the CNGA3 ishuman CNGA3.

In one embodiment, the target nucleic acid sequence encodes a Guaninenucleotide-binding protein G(t) subunit alpha-2 (GNAT-2) polypeptide. Ina related embodiment, the GNAT-2 is mouse GNAT-2. In another relatedembodiment, the GNAT-2 is rat GNAT-2. In still another relatedembodiment, the GNAT-2 is human

In another embodiment, the invention features a mammalian cellcomprising the expression vector of any one of the above aspects andembodiments.

In still another embodiment, the invention features a transgeneexpression cassette comprising the nucleic acid of any of the aboveaspects or embodiments, a nucleic acid selected from the groupconsisting of a CNGB3 nucleic acid, a CNGA3 nucleic acid, and a GNAT2nucleic acid, and minimal regulatory elements. In one embodiment, theinvention features a nucleic acid vector comprising the expressioncassette of any one of the above aspects or embodiments. In a relatedembodiment, the vector is an adeno-associated viral (AAV) vector.

In another embodiment, the invention features a kit comprising theexpression vector of any one of the above aspects or embodiments andinstructions for use.

The invention also features in another embodiment, a method of treatingan eye disease comprising administering to a subject in need thereof theexpression vector of any one of the above aspects or embodiments,thereby treating the subject.

The invention also features in another embodiment, a method of promotingCNGA3 or CNGB3 expression in the cone cells of a subject comprisingadministering to the subject the expression vector of any one of theabove aspects or embodiments, thereby promoting CNGA3 or CNGB3expression.

In one embodiment, the eye disease is associated with a geneticmutation, substitution, or deletion that affects retinal cone cells. Inanother embodiment, the eye disease affects the retinal pigmentepithelium. In another related embodiment, the eye disease isachromatopsia.

In another embodiment, the expression vector is capable of promotingCNGB3 expression in S-cone cells, M-cone cells, and L-cone cells. Inanother further embodiment, the expression vector is capable ofpromoting CNGA3 expression in S-cone cells, M-cone cells, and L-conecells. In still another further embodiment, the expression vector iscapable of promoting GNAT-2 expression in S-cone cells, M-cone cells,and L-cone cells.

In further embodiments, the vector is administered subretinally.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic drawing of the truncated human red/green opsinpromoter.

FIG. 2: Schematic drawing of the rAAV5-PR2.1-hCNGB3 vector.

FIG. 3: Schematic drawings of four proviral plasmids that containvariants of the PR2.1 promoter. The PR2.1 promoter (a truncated humanred/green opsin promoter) was truncated at its 5′-end by 300 bp, 500 bp,and 1,100 bp to create shorter promoters, designated PR1.7, PR1.5, andPR1.1, respectively. A CMV enhancer was added to the 5′ end of the PR1.1to create a hybrid promoter. The 500 bp core promoter (shown in gray)and the locus control region (shown in red) of PR2.1 were left intact ineach of these constructs. Terminal repeats are indicated by the arrows,and the location of SV40 splicing signal sequences is shown.

FIG. 4A sets forth SEQ ID NO 1. FIG. 4B sets forth SEQ ID NO 2. FIG. 4Csets forth SEQ ID NO 3. FIG. 4D sets forth SEQ ID NO 4.

FIG. 5 shows the results of experiments to assess the efficiency andspecificity of PR1.1 and PR1.5 to target cones in mice, using rAAVvectors expressing green fluorescent protein (GFP). PNA is a marker forcone photoreceptors. DAPI is used to identify nuclei.

FIG. 6 shows the results of experiments to assess the efficiency andspecificity of PR1.7 and PR2.1 to target cones in mice, using rAAVvectors expressing green fluorescent protein (GFP). PNA is a marker forcone photoreceptors. DAPI is used to identify nuclei.

FIG. 7 shows the results of fundus autofluorescence imaging (FAF) todetect the presence of green fluorescent protein (GFP) in the non-humanprimate (NHP) eyes received subretinally rAAV2tYF-PR2.1-GFP,rAAV2tYF-PR1.7-GFP, or AAV2tYF-CSP-GFP.

FIG. 8 (A-E) shows GFP expression in NHP retinas 3 months afterinjection of AAV2tYF-GFP vectors. The panels show representative retinalsections from a normal control eye without AAV treatment (panel A), orfrom eyes subretinally injected with AAV2tYF-CSP-GFP (panel B),AAV2tYF-PR2.1-GFP (panel C), or AAV2tYF-PR1.7-GFP (panels D & E) stainedwith DAPI for nuclei (blue) and antibodies to GFP (green), L/M coneopsin (red, panels A, B, C & D) or S cone opsin (red, panel E).

FIG. 9 is a graph that shows levels of message RNA (mRNA) of GFP in NHPretinas 3 months after injection of AAV2tYF-GFP vectors. Message RNA(mRNA) of GFP was determined by qRT-PCR, performed in triplicates at 3different times, and normalized by 18S RNA expression in samples.

DETAILED DESCRIPTION OF THE INVENTION I. Overview and Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

The term “such as” is used herein to mean, and is used interchangeably,with the phrase “such as but not limited to”.

A “subject” or “patient” to be treated by the method of the inventioncan mean either a human or non-human animal. A “nonhuman animal”includes any vertebrate or invertebrate organism.

“Achromatopsia” is a color vision disorder. Symptoms of achromatopsiainclude achromatopia (lack of color perception), amblyopia (reducedvisual acuity), hemeralopia (reduced visual capacity in bright lightaccompanied by photoaversion, meaning a dislike or avoidance of brightlight), nystagmus (uncontrolled oscillatory movement of the eyes), irisoperating abnormalities, and impaired stereovision (inability toperceive three-dimensional aspects of a scene). As used herein, the term“achromatopsia” refers to a form of achromatopsia caused by geneticmutations, substitutions, or deletions.

“Treating” a disease (such as, for example, achromatopsia) meansalleviating, preventing, or delaying the occurrence of at least one signor symptom of the disease.

The asymmetric ends of DNA and RNA strands are called the 5′ (fiveprime) and 3′ (three prime) ends, with the 5′ end having a terminalphosphate group and the 3′ end a terminal hydroxyl group. The five prime(5′) end has the fifth carbon in the sugar-ring of the deoxyribose orribose at its terminus. Nucleic acids are synthesized in vivo in the 5′-to 3′-direction, because the polymerase used to assemble new strandsattaches each new nucleotide to the 3′-hydroxyl (—OH) group via aphosphodiester bond.

A “promoter” is a region of DNA that facilitates the transcription of aparticular gene. As part of the process of transcription, the enzymethat synthesizes RNA, known as RNA polymerase, attaches to the DNA neara gene. Promoters contain specific DNA sequences and response elementsthat provide an initial binding site for RNA polymerase and fortranscription factors that recruit RNA polymerase.

The retina contains three kinds of photoreceptors: rod cells, conecells, and photoreceptive ganglion cells. Cone cells are of three types:S-cone cells, M-cone cells, and L-cone cells. S-cone cells respond moststrongly to short wavelength light (peak near 420-440 nm) and are alsoknown as blue cones. M-cone cells respond most strongly to mediumwavelength light (peak near 534-545 nm) and are also known as greencones. L-cone cells respond most strongly to light of long wavelengths(peak near 564-580 nm) and are also known as red cones. The differencein the signals received from the three cone types allows the brain toperceive all possible colors.

A “transgene expression cassette” or “expression cassette” comprises thegene sequences that a nucleic acid vector is to deliver to target cells.These sequences include the gene of interest (e.g., a CNGB3 or CNGA3nucleic acid), one or more promoters, and minimal regulatory elements.

“Minimal regulatory elements” are regulatory elements that are necessaryfor effective expression of a gene in a target cell and thus should beincluded in a transgene expression cassette. Such sequences couldinclude, for example, promoter or enhancer sequences, a polylinkersequence facilitating the insertion of a DNA fragment within a plasmidvector, and sequences responsible for intron splicing andpolyadenlyation of mRNA transcripts. In a recent example of a genetherapy treatment for achromatopsia, the expression cassette includedthe minimal regulatory elements of a polyadenylation site, splicingsignal sequences, and AAV inverted terminal repeats. See, e.g., Komaromyet al.

A “nucleic acid” or “nucleic acid molecule” is a molecule composed ofchains of monomeric nucleotides, such as, for example, DNA molecules(e.g., cDNA or genomic DNA). A nucleic acid may encode, for example, apromoter, the CNGB3 or CNGA3 gene or portion thereof, or regulatoryelements. A nucleic acid molecule can be single-stranded ordouble-stranded. A “CNGB3 nucleic acid” refers to a nucleic acid thatcomprises the CNGB3 gene or a portion thereof, or a functional variantof the CNGB3 gene or a portion thereof. Similarly, a “CNGA3 nucleicacid” refers to a nucleic acid that comprises the CNGA3 gene or aportion thereof, or a functional variant of the CNGA3 gene or a portionthereof, and a “GNAT2 nucleic acid” refers to a nucleic acid thatcomprises the GNAT2 gene or a portion thereof, or a functional variantof the GNAT2 gene or a portion thereof. A functional variant of a geneincludes a variant of the gene with minor variations such as, forexample, silent mutations, single nucleotide polymorphisms, missensemutations, and other mutations or deletions that do not significantlyalter gene function.

An “isolated” nucleic acid molecule (such as, for example, an isolatedpromoter) is one which is separated from other nucleic acid moleculeswhich are present in the natural source of the nucleic acid. Forexample, with regard to genomic DNA, the term “isolated” includesnucleic acid molecules which are separated from the chromosome withwhich the genomic DNA is naturally associated. Preferably, an “isolated”nucleic acid molecule is free of sequences which naturally flank thenucleic acid molecule in the genomic DNA of the organism from which thenucleic acid molecule is derived.

II. Methods of the Invention

The present invention provides promoters, expression cassettes, vectors,kits, and methods that can be used in the treatment of genetic diseasesthat affect the cone cells of the retina. Genetic diseases that affectthe cone cells of the retina include achromatopsia; Leber congenitalamaurosis; cone-rod dystrophy; retinitis pigmentosa, including X-linkedretinitis pigmentosa; maculopathies; and age-related maculardegeneration. In preferred embodiments, the disease is achromatopsia.

Achromatopsia is a color vision disorder. Autosomal recessive mutationsor other types of sequence alterations in three genes are thepredominant cause of congenital achromatopsia. See Pang, J.-J. et al.(2010). Achromatopsia as a Potential Candidate for Gene Therapy. InAdvances in Experimental Medicine and Biology, Volume 664, Part 6,639-646 (2010). Achromatopsia has been associated with mutations ineither the alpha or beta subunits of cyclic nucleotide gated channels(CNGs), which are respectively known as CNGA3 and CNGB3. Mutations inthe CNGA3 gene that were associated with achromatopsia are reported inPatel K A, et al. Transmembrane S1 mutations in CNGA3 from achromatopsia2 patients cause loss of function and impaired cellular trafficking ofthe cone CNG channel. Invest. Ophthalmol. Vis. Sci. 46 (7): 2282-90.(2005), Johnson S, et al. Achromatopsia caused by novel mutations inboth CNGA3 and CNGB3. J. Med. Genet. 41 (2): e20. (2004), Wissinger B,et al. CNGA3 mutations in hereditary cone photoreceptor disorders. Am.J. Hum. Genet. 69 (4): 722-37. (2001), and Kohl S, et al. Totalcolourblindness is caused by mutations in the gene encoding thealpha-subunit of the cone photoreceptor cGMP-gated cation channel Nat.Genet. 19 (3): 257-9. (1998). Mutations in CNGB3 gene that wereassociated with achromatopsia are reported in Johnson S, et al.Achromatopsia caused by novel mutations in both CNGA3 and CNGB3. J. Med.Genet. 41 (2): e20. (2004), Peng C, et al. Achromatopsia-associatedmutation in the human cone photoreceptor cyclic nucleotide-gated channelCNGB3 subunit alters the ligand sensitivity and pore properties ofheteromeric channels. J. Biol. Chem. 278 (36): 34533-40 (2003), Bright SR, et al. Disease-associated mutations in CNGB3 produce gain of functionalterations in cone cyclic nucleotide-gated channels. Mol. Vis. 11:1141-50 (2005), Kohl S, et al. CNGB3 mutations account for 50% of allcases with autosomal recessive achromatopsia. Eur. J. Hum. Genet. 13(3): 302-8 (2005), Rojas C V, et al. A frameshift insertion in the conecyclic nucleotide gated cation channel causes complete achromatopsia ina consanguineous family from a rural isolate. Eur. J. Hum. Genet. 10(10): 638-42 (2002), Kohl S, et al. Mutations in the CNGB3 gene encodingthe beta-subunit of the cone photoreceptor cGMP-gated channel areresponsible for achromatopsia (ACHM3) linked to chromosome 8q21. Hum.Mol. Genet. 9 (14): 2107-16 (2000), Sundin O H, et al. Genetic basis oftotal colourblindness among the Pingelapese islanders. Nat. Genet. 25(3): 289-93 (2000). Sequence alterations in the gene for cone celltransducin, known as GNAT2, can also cause achromatopsia. See Kohl S, etal., Mutations in the cone photoreceptor G-protein alpha-subunit geneGNAT2 in patients with achromatopsia. Kokl S, et al. Mutations in thecone photoreceptor G-protein alpha-subunit gene GNAT2 in patients withachromatopsia. Am J Hum Genet 71 (2): 422-425 (2002) (hereinafter Kohlet al.). The severity of mutations in these proteins correlates with theseverity of the achromatopsia phenotype.http://en.wikipedia.org/wiki/Achromatopsia. Mutations in CNGB3 accountfor about 50% of cases of achromatopsia. Kohl et al. Mutations in CNGA3account for about 23% of cases, and mutations in GNAT2 account for about2% of cases.

The “CNGB3 gene” is the gene that encodes the cyclic nucleotide-gatedchannel beta 3 (CNGB3). The “CNGA3 gene” is the gene that encodes thecyclic nucleotide-gated channel alpha 3 (CNGA3). The CNGB3 and CNGA3genes are expressed in cone cells of the retina. Native retinal cyclicnucleotide gated channels (CNGs) are critically involoved inphototransduction. CNGs are cation channels that consist of two alphaand two beta subunits. In the dark, cones have a relatively highconcentration of cyclic guanosine 3′-5′ monophosphate (cGMP), whichcauses the CNGs to open, resulting in depolarization and continuousglutamate release. Light exposure activates a signal transductionpathway that breaks down cGMP. The reduction in cGMP concentrarioncauses the CNGs to close, preventing the influx of positive ions,hyperpolarizing the cell, and stopping the release of glutamate.Mutations in either the CNGB3 or CNGA3 genes can cause defects in conephotoreceptor function resulting in achromatopsia. Mutations in theCNGB3 gene have been associated with other diseases in addition toachromatopsia, including progressive cone dystrophy and juvenile maculardegeneration.

The GNAT2 gene encodes the alpha-2 subunit of guanine nucleotide bindingprotein, which is also known as the cone-specific alpha transducin.Guanine nucleotide-binding proteins (G proteins) consist of alpha, beta,and gamma subunits. In photoreceptors, G proteins are critical in theamplification and transduction of visual signals. Various types ofsequence alterations in GNAT2 can cause human achromatopsia: nonsensemutations, small deletion and/or insertion mutations, frameshiftmutations, and large intragenic deletions. Pang et al.

Currently, there is no effective treatment for achromatopsia. Animalstudies suggest that it is possible to use gene therapy to treatachromatopsia and other diseases of the retina. For recessive genedefects, the goal is to deliver a wild-type copy of a defective gene tothe affected retinal cell type. The ability to deliver genes to somesubsets of cone cells was demonstrated, for example, in Mauck, M. C. etal., Longitudinal evaluation of expression of virally deliveredtransgenes in gerbil cone photoreceptors. Visual Neuroscience 25(3):273-282 (2008). The authors showed that a recombinant AAV vector couldbe used to achieve long-term expression of a reporter gene encodinggreen fluorescent protein in specific types of gerbil cone cells. Theauthors further demonstrated that a human long-wavelength opsin genecould be delivered to specific gerbil cones, resulting in cone responsesto long-wavelength light.

Other studies demonstrated that gene therapy with recombinant AAVvectors could be used to convert dichromat monkeys into trichromats byintroducing a human L-opsin gene into the squirrel monkey retina.Mancuso, K., et al. Gene therapy for red-green colour blindness in adultprimates. Nature 461: 784-787 (2009). Electroretinograms verified thatthe introduced photopigment was functional, and the monkeys showedimproved color vision in a behavioral test.

There are several animal models of achromatopsia for which gene therapyexperiments have demonstrated the ability to restore cone function. SeePang et al. First, the Gnat2^(cpfl3) mouse has a recessive mutation inthe cone-specific alpha transducin gene, resulting in poor visual acuityand little or no cone-specific ERT response. Treatment of homozygousGnat2^(cpfl3) mice with a single subretinal injection of an AAV serotype5 vector carrying wild type mouse GNAT2 cDNA and a human red cone opsinpromoter restored cone-specific ERG responses and visual acuity.Alexander et al. Restoration of cone vision in a mouse model ofachromatopsia. Nat Med 13:685-687 (2007) (hereinafter Alexander et al.).Second, the cpfl5 (Cone Photoreceptor Function Loss 5) mouse has anautosomal recessive missense mutation in the CNGA3 gene with nocone-specific ERG response. Treatment of cpfl5 mice with subretinalinjection of an AAV vector carrying the wild type mouse CNGA3 gene and ahuman blue cone promoter (HB570) resulted in restoration ofcone-specific ERG responses. Pang et al. Third, there is an AlaskanMalmute dog that has a naturally occurring CNGB3 mutation causing lossof daytime vision and absence of retinal cone function. In this type ofdog, subretinal injection of an AAV5 vector containing human CNGB3 cDNAand a human red cone opsin promoter restored cone-specific ERGresponses. See, e.g., Komaromy et al.

The prior methods for treatment of achromatopsia using gene therapy werelimited by the fact that the promoters used caused expression oftransgenes only in certain types of cone cell photoreceptors. Thepromoters of the present invention can drive gene expression in allthree types of cone cells that are present in humans (S-cone cells,M-cone cells, and L-cone cells).

Another limitation of the studies performed by Komaromy et al. was thatthe overall size of the expression cassette utilized (5,230 bp) was wellbeyond the normal packaging capacity (<4.9 kb) of AAV particles; theover-stuffed rAAV particles dramatically impaired the rAAV packagingefficiency, resulting in low yields, a higher empty-to-full particleratio, and likely a lower infectivity of the vector. Expressioncassettes containing a shorter version of the cone red opsin promoter,or a cone arrestin promoter, were much less effective in restoringvisual function. The promoters of the present invention have theadvantage that due to their shortened length, they make the hCNGB3expression cassette efficiently package in an AAV particle. A promoterthat fits within the normal rAAV packaging capacity provides benefits,such as improved yields, a lower empty-to-full particle ratio, higherinfectivity of the vector, and ultimately, higher efficacy for treatmentof the desired condition.

III. Promoters, Expression Cassettes, Nucleic Acids, and Vectors of theInvention

The promoters, CNGB3 nucleic acids, regulatory elements, and expressioncassettes, and vectors of the invention may be produced using methodsknown in the art. The methods described below are provided asnon-limiting examples of such methods.

Promoters

The present invention provides isolated and/or truncated promoters. Insome aspects, these promoters include a segment of the PR 2.1 promoter.In one embodiment, the promoter is a truncated PR2.1 promoter.

In some embodiments of the promoters of the invention, the promoter iscapable of promoting expression of a transgene in S-cone, M-cone, andL-cone cells. A “transgene” refers to a segment of DNA containing a genesequence that has been isolated from one organism and is introduced intoa different organism. For example, to treat an individual who hasachromatopsia caused by a mutation of the human CNGB3 gene, a wild-type(i.e., non-mutated, or functional variant) human CNGB3 gene may beadministered using an appropriate vector. The wild-type gene is referredto as a “transgene.” In preferred embodiments, the transgene is awild-type version of a gene that encodes a protein that is normallyexpressed in cone cells of the retina. In one such embodiment, thetransgene is derived from a human gene. In a first specific embodiment,the promoter is capable of promoting expression of a CNGB3 nucleic acidin S-cone, M-cone, and L-cone cells. In a second specific embodiment,the promoter is capable of promoting expression of a CNGA3 nucleic acidin S-cone, M-cone, and L-cone cells. In a third specific embodiment, thepromoter is capable of promoting expression of a GNAT2 nucleic acid inS-cone, M-cone, and L-cone cells. In these three specific embodiments,the CNGB3, CNGA3, or GNAT2 is preferably human CNGB3, CNGA3, or GNAT2.

In another aspect, the present invention provides promoters that areshortened versions of the PR2.1 promoter. Such promoters have theadvantage that they fit better within the packaging capacity of AAVparticles and therefore provide advantages such as, for example,improved yields, a lower empty-to-full particle ratio, and higherinfectivity of the vector. In some embodiments, these promoters arecreated by truncating the 5′-end of PR2.1 or the 3′-end of PR 2.1. Insome such embodiments, the lengths of the truncations are selected fromthe group consisting of approximately 300 bp, 500 bp, and 1,100 bp (see,e.g., PR1.7, PR1.5, and PR1.1, respectively).

Expression Cassettes

In another aspect, the present invention provides a transgene expressioncassette that includes (a) a promoter of the invention; (b) a nucleicacid selected from the group consisting of a CNGB3 nucleic acid, a CNGA3nucleic acid, and a GNAT2 nucleic acid; and (c) minimal regulatoryelements. A promoter of the invention includes the promoters discussedsupra.

A “CNGB3 nucleic acid” refers to a nucleic acid that comprises the CNGB3gene or a portion thereof, or a functional variant of the CNGB3 gene ora portion thereof. Similarly, a “CNGA3 nucleic acid” refers to a nucleicacid that comprises the CNGA3 gene or a portion thereof, or a functionalvariant of the CNGA3 gene or a portion thereof, and a “GNAT2 nucleicacid” refers to a nucleic acid that comprises the GNAT2 gene or aportion thereof, or a functional variant of the GNAT2 gene or a portionthereof. A functional variant of a gene includes a variant of the genewith minor variations such as, for example, silent mutations, singlenucleotide polymorphisms, missense mutations, and other mutations ordeletions that do not significantly alter gene function.

In certain embodiments, the nucleic acid is a human nucleic acid (i.e.,a nucleic acid that is derived from a human CNGB3, CNGA3, or GNAT2gene). In other embodiments, the nucleic acid is a non-human nucleicacid (i.e., a nucleic acid that is derived from a non-human CNGB3,CNGA3, or GNAT2 gene).

“Minimal regulatory elements” are regulatory elements that are necessaryfor effective expression of a gene in a target cell. Such regulatoryelements could include, for example, promoter or enhancer sequences, apolylinker sequence facilitating the insertion of a DNA fragment withina plasmid vector, and sequences responsible for intron splicing andpolyadenlyation of mRNA transcripts. In a recent example of a genetherapy treatment for achromatopsia, the expression cassette includedthe minimal regulatory elements of a polyadenylation site, splicingsignal sequences, and AAV inverted terminal repeats. See, e.g., Komaromyet al. The expression cassettes of the invention may also optionallyinclude additional regulatory elements that are not necessary foreffective incorporation of a gene into a target cell.

Vectors

The present invention also provides vectors that include any one of theexpression cassettes discussed in the preceding section. In someembodiments, the vector is an oligonucleotide that comprises thesequences of the expression cassette. In specific embodiments, deliveryof the oligonucleotide may be accomplished by in vivo electroporation(see, e.g., Chalberg, T W, et al. phiC31 integrase confers genomicintegration and long-term transgene expression in rat retina.Investigative Ophthalmology & Visual Science, 46, 2140-2146 (2005)(hereinafter Chalberg et al., 2005)) or electron avalanche transfection(see, e.g., Chalberg, T W, et al. Gene transfer to rabbit retina withelectron avalanche transfection. Investigative Ophthalmology & VisualScience, 47, 4083-4090 (2006) (hereinafter Chalberg et al., 2006)). Infurther embodiments, the vector is a DNA-compacting peptide (see, e.g.,Farjo, R, et al. Efficient non-viral ocular gene transfer with compactedDNA nanoparticles. PLoS ONE, 1, e38 (2006) (hereinafter Farjo et al.,2006), where CK30, a peptide containing a cystein residue coupled topolyethylene glycol followed by 30 lysines, was used for gene transferto photoreceptors), a peptide with cell penetrating properties (seeJohnson, L N, et al., Cell-penetrating peptide for enhanced delivery ofnucleic acids and drugs to ocular tissues including retina and cornea.Molecular Therapy, 16(1), 107-114 (2007) (hereinafter Johnson et al.,2007), Barnett, E M, et al. Selective cell uptake of modified Tatpeptide-fluorophore conjugates in rat retina in ex vivo and in vivomodels. Investigative Ophthalmology & Visual Science, 47, 2589-2595(2006) (hereinafter Barnett et al., 2006), Cashman, S M, et al. Evidenceof protein transduction but not intercellular transport by proteinsfused to HIV tat in retinal cell culture and in vivo. Molecular Therapy,8, 130-142 (2003) (hereinafter Cashman et al., 2003), Schorderet, D F,et al. D-TAT transporter as an ocular peptide delivery system. Clinicaland Experimental Ophthalmology, 33, 628-635 (2005)(hereinafterSchorderet et al., 2005), Kretz, A, et al. HSV-1 VP22 augmentsadenoviral gene transfer to CNS neurons in the retina and striatum invivo. Molecular Therapy, 7, 659-669 (2003)(hereinafter Kretz et al.2003) for examples of peptide delivery to ocular cells), or aDNA-encapsulating lipoplex, polyplex, liposome, or immunoliposome (seee.g., Zhang, Y, et al. Organ-specific gene expression in the rhesusmonkey eye following intravenous nonviral gene transfer. MolecularVision, 9, 465-472 (2003) (hereinafter Zhang et al. 2003), Zhu, C, etal. Widespread expression of an exogenous gene in the eye afterintravenous administration. Investigative Ophthalmology & VisualScience, 43, 3075-3080 (2002) (hereinafter Zhu et al. 2002), Zhu, C., etal. Organ-specific expression of the lacZ gene controlled by the opsinpromoter after intravenous gene administration in adult mice. Journal ofGene Medicine, 6, 906-912. (2004) (hereinafter Zhu et al. 2004)).

In preferred embodiments, the vector is a viral vector, such as a vectorderived from an adeno-associated virus, an adenovirus, a retrovirus, alentivirus, a vaccinia/poxvirus, or a herpesvirus (e.g., herpes simplexvirus (HSV)). See e.g., Howarth. In the most preferred embodiments, thevector is an adeno-associated viral (AAV) vector.

Multiple serotypes of adeno-associated virus (AAV), including 12 humanserotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, and AAV12) and more than 100 serotypes from nonhuman primateshave now been identified. Howarth J L et al., Using viral vectors asgene transfer tools. Cell Biol Toxicol 26:1-10 (2010) (hereinafterHowarth et al.). In embodiments of the present invention wherein thevector is an AAV vector, the serotype of the inverted terminal repeats(ITRs) of the AAV vector may be selected from any known human ornonhuman AAV serotype. In preferred embodiments, the serotype of the AAVITRs of the AAV vector is selected from the group consisting of AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.Moreover, in embodiments of the present invention wherein the vector isan AAV vector, the serotype of the capsid sequence of the AAV vector maybe selected from any known human or animal AAV serotype. In someembodiments, the serotype of the capsid sequence of the AAV vector isselected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In preferredembodiments, the serotype of the capsid sequence is AAV5. In someembodiments wherein the vector is an AAV vector, a pseudotyping approachis employed, wherein the genome of one ITR serotype is packaged into adifferent serotype capsid. See e.g., Zolutuhkin S. et al. Production andpurification of serotype 1, 2, and 5 recombinant adeno-associated viralvectors. Methods 28(2): 158-67 (2002). In preferred embodiments, theserotype of the AAV ITRs of the AAV vector and the serotype of thecapsid sequence of the AAV vector are independently selected from thegroup consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, and AAV12.

In some embodiments of the present invention wherein the vector is arAAV vector, a mutant capsid sequence is employed. Mutant capsidsequences, as well as other techniques such as rational mutagenesis,engineering of targeting peptides, generation of chimeric particles,library and directed evolution approaches, and immune evasionmodifications, may be employed in the present invention to optimize AAVvectors, for purposes such as achieving immune evasion and enhancedtherapeutic output. See e.g., Mitchell A. M. et al. AAV's anatomy:Roadmap for optimizing vectors for translational success. Curr GeneTher. 10(5): 319-340.

Making the Nucleic Acids of the Invention

A nucleic acid molecule (including, for example, a promoter, CNGB3nucleic acid, CNGA3 nucleic acid, a GNAT2 nucleic acid, or a regulatoryelement) of the present invention can be isolated using standardmolecular biology techniques. Using all or a portion of a nucleic acidsequence of interest as a hybridization probe, nucleic acid moleculescan be isolated using standard hybridization and cloning techniques(e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning. A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989).

A nucleic acid molecule for use in the methods of the invention can alsobe isolated by the polymerase chain reaction (PCR) using syntheticoligonucleotide primers designed based upon the sequence of a nucleicacid molecule of interest. A nucleic acid molecule used in the methodsof the invention can be amplified using cDNA, mRNA or, alternatively,genomic DNA as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques.

Furthermore, oligonucleotides corresponding to nucleotide sequences ofinterest can also be chemically synthesized using standard techniques.Numerous methods of chemically synthesizing polydeoxynucleotides areknown, including solid-phase synthesis which has been automated incommercially available DNA synthesizers (See e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071, incorporated byreference herein). Automated methods for designing syntheticoligonucleotides are available. See e.g., Hoover, D. M. & Lubowski, J.Nucleic Acids Research, 30(10): e43 (2002).

Many embodiments of the invention involve a CNGB3 nucleic acid, a CNGA3nucleic acid, or a GNAT2 nucleic acid. Some aspects and embodiments ofthe invention involve other nucleic acids, such as isolated promoters orregulatory elements. A nucleic acid may be, for example, a cDNA or achemically synthesized nucleic acid. A cDNA can be obtained, forexample, by amplification using the polymerase chain reaction (PCR) orby screening an appropriate cDNA library. Alternatively, a nucleic acidmay be chemically synthesized.

IV. Methods and Kits of the Invention Methods of Treatment

The invention provides methods for treating a disease associated with agenetic mutation, substitution, or deletion that affects retinal conecells, wherein the methods comprise administering to a subject in needof such treatment a vector that includes one of the promoters of theinvention, thereby treating the subject. In a preferred embodiment, thedisease is achromatopsia. Other diseases associated with a geneticmutation, substitution, or deletion that affects retinal cone cellsinclude achromatopsia, Leber congenital amaurosis, cone-rod dystrophy,maculopathies, age-related macular degeneration and retinitispigmentosa, including X-linked retinitis pigmentosa.

The invention further provides methods for treating achromatopsiacomprising administering any of the vectors of the invention to asubject in need of such treatment, thereby treating the subject.

A “subject” to be treated by the methods of the invention can meaneither a human or non-human animal. A “nonhuman animal” includes anyvertebrate or invertebrate organism. In some embodiments, the nonhumananimal is an animal model of retinal disease, or of achromatopsia inparticular. See e.g., Pang et al., Alexander et al., Komaromy et al.Various large animal models are available for the study of AAV-mediatedgene-based therapies in the retina. Stieger K. et al. AAV-mediated genetherapy for retinal disorders inlarge animal models. ILAR J. 50(2):206-224 (2009). The promoters of the invention are described supra.“Treating” a disease (such as, for example, achromatopsia) meansalleviating, preventing, or delaying the occurrence of at least one signor symptom of the disease. A “sign” of a disease is a manifestation ofthe disease that can be observed by others or measured by objectivemethods, such as, e.g., electroretinography or behavioral testing. A“symptom” of a disease is a characteristic of the disease that issubjectively perceived by the subject.

In either of these two methods of treatment, the vector can be any typeof vector known in the art. In some embodiments, the vector is anon-viral vector, such as a naked DNA plasmid, an oligonucleotide (suchas, e.g., an antisense oligonucleotide, a small molecule RNA (siRNA), adouble stranded oligodeoxynucleotide, or a single stranded DNAoligonucleotide). In specific embodiments involving oligonucleotidevectors, delivery may be accomplished by in vivo electroporation (seee.g., Chalberg et al., 2005) or electron avalanche transfection (seee.g., Chalberg et al. 2006). In further embodiments, the vector is adendrimer/DNA complex that may optionally be encapsulated in a watersoluble polymer, a DNA-compacting peptide (see e.g., Farjo et al. 2006,where CK30, a peptide containing a cystein residue coupled to polyethylene glycol followed by 30 lysines, was used for gene transfer tophotoreceptors), a peptide with cell penetrating properties (see Johnsonet al. 2007; Barnett et al., 2006; Cashman et al., 2003; Schorder etal., 2005; Kretz et al. 2003 for examples of peptide delivery to ocularcells), or a DNA-encapsulating lipoplex, polyplex, liposome, orimmunoliposome (see e.g., Zhang et al. 2003; Zhu et al. 2002; Zhu et al.2004). In many additional embodiments, the vector is a viral vector,such as a vector derived from an adeno-associated virus, an adenovirus,a retrovirus, a lentivirus, a vaccinia/poxvirus, or a herpesvirus (e.g.,herpes simplex virus (HSV)). See e.g., Howarth. In preferredembodiments, the vector is an adeno-associated viral (AAV) vector.

In the methods of treatment of the present invention, administering of avector can be accomplished by any means known in the art. In preferredembodiments, the administration is by subretinal injection. In certainembodiments, the subretinal injection is delivered preferentially to oneor more regions where cone density is particularly high (such as e.g.,the tapetal zone superior to the optic disc). In other embodiments, theadministration is by intraocular injection, intravitreal injection, orintravenous injection. Administration of a vector to the retina may beunilateral or bilateral and may be accomplished with or without the useof general anesthesia.

In the methods of treatment of the present invention, the volume ofvector delivered may be determined based on the characteristics of thesubject receiving the treatment, such as the age of the subject and thevolume of the area to which the vector is to be delivered. It is knownthat eye size and the volume of the subretinal space differ amongindividuals and may change with the age of the subject. In embodimentswherein the vector is administered subretinally, vector volumes may bechosen with the aim of covering all or a certain percentage of thesubretinal space, or so that a particular number of vector genomes isdelivered.

In the methods of treatment of the present invention, the concentrationof vector that is administered may differ depending on production methodand may be chosen or optimized based on concentrations determined to betherapeutically effective for the particular route of administration. Insome embodiments, the concentration in vector genomes per milliliter(vg/ml) is selected from the group consisting of about 10⁸ vg/ml, about10⁹ vg/ml, about 10¹⁰ vg/ml, about 10¹¹ vg/ml, about 10¹² vg/ml, about10¹³ vg/ml, and about 10¹⁴ vg/ml. In preferred embodiments, theconcentration is in the range of 10¹⁰ vg/ml-10¹³ vg/ml, delivered bysubretinal injection or intravitreal injection in a volume of about 0.1mL, about 0.2 mL, about 0.4 mL, about 0.6 mL, about 0.8 mL, and about1.0 mL

Kits

The present invention also provides kits. In one aspect, a kit of theinvention comprises a vector that comprises (a) any one of the promotersof the invention and (b) instructions for use thereof. In anotheraspect, a kit of the invention comprises (a) any one of the vectors ofthe invention, and (b) instructions for use thereof. The promoters andvectors of the invention are described supra. In some embodiments, avector of the invention may be any type of vector known in the art,including a non-viral or viral vector, as described supra. In preferredembodiments, the vector is a viral vector, such as a vector derived froman adeno-associated virus, an adenovirus, a retrovirus, a lentivirus, avaccinia/poxvirus, or a herpesvirus (e.g., herpes simplex virus (HSV)).In the most preferred embodiments, the vector is an adeno-associatedviral (AAV) vector.

The instructions provided with the kit may describe how the promoter canbe incorporated into a vector or how the vector can be administered fortherapeutic purposes, e.g., for treating a disease associated with agenetic mutation, substitution, or deletion that affects retinal conecells. In some embodiments wherein the kit is to be used for therapeuticpurposes, the instructions include details regarding recommended dosagesand routes of administration.

Methods of Making Recombinant Adeno-Associated Viral Vectors (AAVVectors)

The present invention also provides methods of making a recombinantadeno-associated viral (rAAV) vector comprising inserting into anadeno-associated viral vector any one of the promoters of the invention(described supra) and a nucleic acid selected from the group consistingof a CNGB3 nucleic acid, a CNGA3 nucleic acid, and a GNAT2 nucleic acid(also described supra). In some embodiments, the nucleic acid is a humannucleic acid, i.e., a nucleic acid derived from a human CNGB3, CNGA orGNAT gene, or a functional variant thereof. In alternative embodiments,the nucleic acid is a nucleic acid derived from a non-human gene.

In the methods of making an rAAV vector that are provided by theinvention, the serotype of the capsid sequence and the serotype of theITRs of said AAV vector are independently selected from the groupconsisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV10, AAV11, and AAV12. Thus, the invention encompasses vectors thatuse a pseudotyping approach, wherein the genomne of one ITR serotype ispackaged into a different serotype capsid. See e.g., Daya S. and Berns,K. I., Gene therapy using adeno-associated virus vectors. ClinicalMicrobiology Reviews, 21(4): 583-593 (2008) (hereinafter Daya et al.).Furthermore, in some embodiments, the capsid sequence is a mutant capsidsequence.

AAV Vectors

AAV vectors are derived from adeno-associated virus, which has its namebecause it was originally described as a contaminant of adenoviruspreparations. AAV vectors offer numerous well-known advantages overother types of vectors: wildtype strains infect humans and nonhumanprimates without evidence of disease or adverse effects; the AAV capsiddisplays very low immunogenicity combined with high chemical andphysical stability which permits rigorous methods of virus purificationand concentration; AAV vector transduction leads to sustained transgeneexpression in post-mitotic, nondividing cells and provides long-termgain of function; and the variety of AAV subtypes and variants offersthe possibility to target selected tissues and cell types. Heilbronn R &Weger S, Viral Vectors for Gene Transfer: Current Status of GeneTherapeutics, in M. Schafer-Korting (ed.), Drug Delivery, Handbook ofExperimental Pharmacology, 197: 143-170 (2010) (hereinafter Heilbronn).A major limitation of AAV vectors is that the AAV offers only a limitedtransgene capacity (<4.9 kb) for a conventional vector containingsingle-stranded DNA.

AAV is a nonenveloped, small, single-stranded DNA-containing virusencapsidated by an icosahedral, 20 nm diameter capsid. The humanserotype AAV2 was used in a majority of early studies of AAV. Heilbronn.It contains a 4.7 kb linear, single-stranded DNA genome with two openreading frames rep and cap (“rep” for replication and “cap” for capsid).Rep codes for four overlapping nonstructural proteins: Rep78, Rep68,Rep52, and Rep40. Rep78 and Rep69 are required for most steps of the AAVlife cycle, including the initiation of AAV DNA replication at thehairpin-structured inverted terminal repeats (ITRs), which is anessential step for AAV vector production. The cap gene codes for threecapsid proteins, VP1, VP2, and VP3. Rep and cap are flanked by 145 bpITRs. The ITRs contain the origins of DNA replication and the packagingsignals, and they serve to mediate chromosomal integration. The ITRs aregenerally the only AAV elements maintained in AAV vector construction.

To achieve replication, AAVs must be coinfected into the target cellwith a helper virus. Grieger J C & Samulski R J, Adeno-associated virusas a gene therapy vector: Vector development, production, and clinicalapplications. Adv Biochem Engin/Biotechnol 99:119-145 (2005). Typically,helper viruses are either adenovirus (Ad) or herpes simplex virus (HSV).In the absence of a helper virus, AAV can establish a latent infectionby integrating into a site on human chromosome 19. Ad or HSV infectionof cells latently infected with AAV will rescue the integrated genomeand begin a productive infection. The four Ad proteins required forhelper function are E1A, E1B, E4, and E2A. In addition, synthesis of Advirus-associated (VA) RNAs is required. Herpesviruses can also serve ashelper viruses for productive AAV replication. Genes encoding thehelicase-primase complex (UL5, UL8, and UL52) and the DNA-bindingprotein (UL29) have been found sufficient to mediate the HSV helpereffect. In some embodiments of the present invention that employ rAAVvectors, the helper virus is an adenovirus. In other embodiments thatemploy rAAV vectors, the helper virus is HSV.

Making Recombinant AAV (rAAV) Vectors

The production, purification, and characterization of the rAAV vectorsof the present invention may be carried out using any of the manymethods known in the art. For reviews of laboratory-scale productionmethods, see, e.g., Clark R K, Recent advances in recombinantadeno-associated virus vector production. Kidney Int. 61s:9-15 (2002);Choi V W et al., Production of recombinant adeno-associated viralvectors for in vitro and in vivo use. Current Protocols in MolecularBiology 16.25.1-16.25.24 (2007) (hereinafter Choi et al.); Grieger J C &Samulski R J, Adeno-associated virus as a gene therapy vector: Vectordevelopment, production, and clinical applications. Adv BiochemEngin/Biotechnol 99:119-145 (2005) (hereinafter Grieger & Samulski);Heilbronn R & Weger S, Viral Vectors for Gene Transfer: Current Statusof Gene Therapeutics, in M. Schäfer-Korting (ed.), Drug Delivery,Handbook of Experimental Pharmacology, 197: 143-170 (2010) (hereinafterHeilbronn); Howarth J L et al., Using viral vectors as gene transfertools. Cell Biol Toxicol 26:1-10 (2010) (hereinafter Howarth). Theproduction methods described below are intended as non-limitingexamples.

AAV vector production may be accomplished by cotransfection of packagingplasmids. Heilbronn. The cell line supplies the deleted AAV genes repand cap and the required helpervirus functions. The adenovirus helpergenes, VA-RNA, E2A and E4 are transfected together with the AAV rep andcap genes, either on two separate plasmids or on a single helperconstruct. A recombinant AAV vector plasmid wherein the AAV capsid genesare replaced with a transgene expression cassette (comprising the geneof interest, e.g., a CNGB3 nucleic acid; a promoter; and minimalregulatory elements) bracketed by ITRs, is also transfected. Thesepackaging plasmids are typically transfected into 293 cells, a humancell line that constitutively expresses the remaining required Ad helpergenes, E1A and E1B. This leads to amplification and packaging of the AAVvector carrying the gene of interest.

Multiple serotypes of AAV, including 12 human serotypes and more than100 serotypes from nonhuman primates have now been identified. Howarthet al. The AAV vectors of the present invention may comprise capsidsequences derived from AAVs of any known serotype. As used herein, a“known serotype” encompasses capsid mutants that can be produced usingmethods known in the art. Such methods, include, for example, geneticmanipulation of the viral capsid sequence, domain swapping of exposedsurfaces of the capsid regions of different serotypes, and generation ofAAV chimeras using techniques such as marker rescue. See Bowles et al.Marker rescue of adeno-associated virus (AAV) capsid mutants: A novelapproach for chimeric AAV production. Journal of Virology, 77(1):423-432 (2003), as well as references cited therein. Moreover, the AAVvectors of the present invention may comprise ITRs derived from AAVs ofany known serotype. Preferentially, the ITRs are derived from one of thehuman serotypes AAV1-AAV12. In some embodiments of the presentinvention, a pseudotyping approach is employed, wherein the genome ofone ITR serotype is packaged into a different serotype capsid.

Preferentially, the capsid sequences employed in the present inventionare derived from one of the human serotypes AAV1-AAV12. Recombinant AAVvectors containing an AAV5 serotype capsid sequence have beendemonstrated to target retinal cells in vivo. See, for example, Komaromyet al. Therefore, in preferred embodiments of the present invention, theserotype of the capsid sequence of the AAV vector is AAV5. In otherembodiments, the serotype of the capsid sequence of the AAV vector isAAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV5, AAV5, AAV10, AAV11, or AAV12.Even when the serotype of the capsid sequence does not naturally targetretinal cells, other methods of specific tissue targeting may beemployed. See Howarth et al. For example, recombinant AAV vectors can bedirectly targeted by genetic manipulation of the viral capsid sequence,particularly in the looped out region of the AAV three-dimensionalstructure, or by domain swapping of exposed surfaces of the capsidregions of different serotypes, or by generation of AAV chimeras usingtechniques such as marker rescue. See Bowles et al. Marker rescue ofadeno-associated virus (AAV) capsid mutants: A novel approach forchimeric AAV production. Journal of Virology, 77(1): 423-432 (2003), aswell as references cited therein.

One possible protocol for the production, purification, andcharacterization of recombinant AAV (rAAV) vectors is provided in Choiet al. Generally, the following steps are involved: design a transgeneexpression cassette, design a capsid sequence for targeting a specificreceptor, generate adenovirus-free rAAV vectors, purify and titer. Thesesteps are summarized below and described in detail in Choi et al.

The transgene expression cassette may be a single-stranded AAV (ssAAV)vector or a “dimeric” or self-complementary AAV (scAAV) vector that ispackaged as a pseudo-double-stranded transgene. Choi et al.; Heilbronn;Howarth. Using a traditional ssAAV vector generally results in a slowonset of gene expression (from days to weeks until a plateau oftransgene expression is reached) due to the required conversion ofsingle-stranded AAV DNA into double-stranded DNA. In contrast, scAAVvectors show an onset of gene expression within hours that plateauswithin days after transduction of quiescent cells. Heilbronn. However,the packaging capacity of scAAV vectors is approximately half that oftraditional ssAAV vectors. Choi et al. Alternatively, the transgeneexpression cassette may be split between two AAV vectors, which allowsdelivery of a longer construct. See e.g., Daya et al. A ssAAV vector canbe constructed by digesting an appropriate plasmid (such as, forexample, a plasmid containing the hCNGB3 gene) with restrictionendonucleases to remove the rep and cap fragments, and gel purifying theplasmid backbone containing the AAVwt-ITRs. Choi et al. Subsequently,the desired transgene expression cassette can be inserted between theappropriate restriction sites to construct the single-stranded rAAVvector plasmid. A scAAV vector can be constructed as described in Choiet al.

Then, a large-scale plasmid preparation (at least 1 mg) of the rAAVvector and the suitable AAV helper plasmid and pXX6 Ad helper plasmidcan be purified by double CsCl gradient fractionation. Choi et al. Asuitable AAV helper plasmid may be selected from the pXR series,pXR1-pXR5, which respectively permit cross-packaging of AAV2 ITR genomesinto capsids of AAV serotypes 1 to 5. The appropriate capsid may bechosen based on the efficiency of the capsid's targeting of the cells ofinterest. For example, in a preferred embodiment of the presentinvention, the serotype of the capsid sequence of the rAAV vector isAAV5, because this type of capsid is known to effectively target retinalcells. Known methods of varying genome (i.e., transgene expressioncassette) length and AAV capsids may be employed to improve expressionand/or gene transfer to specific cell types (e.g., retinal cone cells).See, e.g., Yang G S, Virus-mediated transduction of murine retina withadeno-associated virus: Effects of viral capsid and genome size. Journalof Virology, 76(15): 7651-7660.

Next, 293 cells are transfected with pXX6 helper plasmid, rAAV vectorplasmid, and AAV helper plasmid. Choi et al. Subsequently thefractionated cell lysates are subjected to a multistep process of rAAVpurification, followed by either CsCl gradient purification or heparinsepharose column purification. The production and quantitation of rAAVvirions may be determined using a dot-blot assay. In vitro transductionof rAAV in cell culture can be used to verify the infectivity of thevirus and functionality of the expression cassette.

In addition to the methods described in Choi et al, various othertransfection methods for production of AAV may be used in the context ofthe present invention. For example, transient transfection methods areavailable, including methods that rely on a calcium phosphateprecipitation protocol.

In addition to the laboratory-scale methods for producing rAAV vectors,the present invention may utilize techniques known in the art forbioreactor-scale manufacturing of AAV vectors, including, for example,Heilbronn; Clement, N. et al. Large-scale adeno-associated viral vectorproduction using a herpesvirus-based system enables manufacturing forclinical studies. Human Gene Therapy, 20: 796-606.

The present invention is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application, as well as the Figures, are expresslyincorporated herein by reference in their entirety.

EXAMPLES Example 1: Creation and Testing of Shorter Versions of thePR2.1 Promoter Materials and Methods

FIG. 2 shows a schematic drawing of the proviral plasmid containing AAVterminal repeats (TR), the PR2.1 promoter and the hCMGB3 transgene. ThePR2.1 promoter was shortened by making truncations starting from the5′-end of PR2.1. The 500 bp core promoter and the 600 bp locus controlregion (LCR) of PR2.1 were left intact. Three shortened versions of thePR2.1 promoter were created: PR1.7, PR1.5, and PR1.1. PR1.7, PR1.5, andPR1.1 were created by truncating PR2.1 at the 5′-end by approximately300 bp, 500 bp, and 1,100 bp, respectively.

SEQ ID NO: 1 corresponds to PR1.1 promoter

SEQ ID NO: 2 corresponds to PR1.5 promoter

SEQ ID NO: 3 corresponds to PR1.7 promoter

SEQ ID NO: 4 corresponds to PR2.1 promoter

A CMV enhancer was added to the 5′ end of the PR1.1 to create a hybridpromoter. Proviral plasmids that contained each of these promoters werecreated, as shown in FIG. 3. These proviral plasmids (p) contained AAVterminal repeats (TR), a synthesized promoter (PR2.1-syn) or truncationsthereof, with or without a CMV enhancer (CMVenh), and a greenfluorescent protein (GFP) transgene. The following four proviralplasmids were constructed and sequenced:

(1) pTR-PR2.1syn-GFP(2) pTR-PR1.8-GFP(3) pTR-PR1.6-GFP(4) pTR-CMVenh-PR1.1-GFP.

To construct pTR-PR2.1syn-GFP, a parental plasmid pTR-CMVenh-hGFP wasfirst constructed from pTR-CBA-hRS1 by replacing the CBA and hRS1sequences with hGFP sequences. The human GFP (hGFP) DNA sequence was PCRamplified from the source with oligonucleotide primers with endonucleaserestriction sites at both ends (Not I and BspHI), digested with NotI/BspHI, and joined into pTR-CBA-hRS1 plasmid that had been digestedwith NotI/NcoI to remove all uncessary DNA sequences including thechicken beta actin promoter and the hRS1 (but not the CMV enhancer). Theresulting plasmid pTR-CMVenh-hGFP contains the CMV enhancer, the hGFPopen reading frame (ORF), and the SV40 poly (A) sequence flanked by AAV2ITRs. The PR2.1 DNA sequence was synthesized according to the DNAsequence 5′ of the human red cone opsin (Wang Y. et al., A locus controlregion adjacent to the human red and green visual pigment genes, Neuron,vol 9, pp 429-440, 1992). The synthesized PR2.1 was composed of basesspanning −4564 to −3009 joined to bases −496 to 0 and contained a LCRessential for expression of both the L and M opsin genes in humans(Komaromy A M et al., Targeting gene expression to cones with human coneopsin promoters in recombinant AAV, Gene Therapy, vol 15, pp 1049-1055,2008). In addition, a 97 base pair SV40 splice donor/splice acceptor(SD/SA) was attached to the end of PR2.1 promoter. Synthesized PR2.1including the SD/SA sequence was inserted into the pJ206 cloning vectorto generate pJ206-PR2.1syn. The PR2.1syn DNA sequence, including theSV40 SD/SA sequence, was released from pJ206-PR2.1syn by HindIII/Acc65Idigestion and inserted into pTR-CMVenh-hGFP that had been digested withHindIII/Acc65I to remove the unnecessary CMV enhancer sequence togenerate the plasmid pTR-PR2.1syn-hGFP.

To construct plasmids with shorter versions of the PR2.1 promoter, thePR2.1 sequence with truncation of 300 bp, 500 bp or 1,100 bp from the 5′end of PR2.1 were PCR amplified from pJ206-PR2.1syn. Fouroligonucleotide primers were designed:

1) PR right-Hind: 5′-GATTTAAGCTTGCGGCCGCGGGTACAATTCCGCAGCTTTTAGAG-3′;2) PR1.1 Left-Hind: 5′-CTGCAAGCTTGTGGGACCACAAATCAG-3′;3) PR1.5 Left-Acc65I: 5′-TAGCGGTACCAGCCATCGGCTGTTAG-3′; and4) PR1.7 left-Acc65I: 5′-GTGGGTACCGGAGGCTGAGGGGTG-3′.Primer PR right-Hind was paired with the other three primers to PCRamplify PR1.1, PR1.5, and PR1.7 respectively. Pfu Ultra HS polymerasemix was used with a thermal cycle of 95° C. for 5 min, and then 35cycles of 94° C. for 1 min, 58° C. for 45 sec, and 72° C. for 2 min.

PR1.1 was amplified from pJ206-PR2.1syn using the primer set of PRright-Hind and PR1.1-left-Hind. The amplified DNA was digested withHindIII and inserted into pTR-CMVenh-hGFP that had been digested withHindIII to generate plasmid pTR-CMVenh-PR1.1-hGFP.

PR1.5 was amplified from pJ206-PR2.1syn using the primer set of PRright-Hind and PR1.5-left-Acc65I. The amplified DNA was digested withHindIII/Acc65I, and inserted into pTR-CMVenh-hGFP that had been digestedwith HindIII/Acc65I to generate plasmid pTR-PR1.5-hGFP

PR1.7 was amplified from pJ206-PR2.1syn using the primer set of PRright-Hind and PR1.7-left-Acc65I. The amplified DNA was digested withHindIII/Acc65I, and inserted into pTR-CMVenh-hGFP that had been digestedwith HindIII/Acc65I to generate plasmid pTR-PR1.7-hGFP.

The DNA sequence of the expression cassette, including the promoter andhGFP, were confirmed by DNA sequencing, and the location of TRs wasconfirmed by SmaI restriction mapping.

To examine if the PR2.1 promoter is functional for RNA transcription andsubesequent protein expression, a human retinal pigment epithelia (RPE)cell line, APRE-19, and human embryonic kidney HEK293 cells were seededin 6-well plates (5×10⁵ cells/well) and then transfected with 1 μg ofDNA from each of six plasmids: pTR-CMVenh-PR1.1-GFP, pTR-PR1.5-GFP,pTR-PR1.7-GFP, pTR-PR2.1syn-GFP, pTR-PR2.1-GFP (Control), orpTR-smCBA-GFP (positive control). Transfected cells were incubated at37° C., 5% CO2 incubator for 4 days. During the period of incubation,transfected cells were examined by fluoresecence microscopy for GFPexpression.

Results

DNA sequencing and restriction mapping of all four plasmids confirmedthat the sequence and the TRs of these proviral plasmids are correct.

In vitro analysis using ARPE-19 and HEK293 cells found that neither ofthese cell lines supported functionality of the PR2.1 promoter. At 24 hpost transfection, strong GFP-expression was observed in cellstransfected with DNA from pTR-smCBA-GFP (positive control). At 48 h posttransfection, weak GFP expression was observed in cells transfected withDNA from pTR-CMVenh-PR1.1-GFP. No GFP-expressing cells were observed inall other wells, i.e. those transfected with DNA from pTR-PR1.5-GFP,pTR-PR1.7-GFP, pTR-PR2.1syn-GFP, or pTR-PR2.1-GFP. Plasmid pTR-PR2.1-GFPcontains the full-length PR2.1 promoter that is known to be functionalfor RNA transcription and subsequent GFP expression in vivo (Komaromy AM et al., Targeting gene expression to cones with human cone opsinpromoters in recombinant AAV, Gene Therapy, vol 15, pp 1049-1055, 2008).Therefore these results indicate that the ARPE-19 cell line does notsupport PR2.1 promotor, neither any other shorter versions of PR2.1promoter. Weak expression of GFP from pTR-CMVenh-PR1.1-GFP transfectedcells is most likely due to the CMV enhancer, which greatly elevates thestrength of the PR1.1 promoter.

Further studies were carried out to evaluate the efficiency andspecificity of PR1.1, PR1.5 PR1.7 and PR2.1 to target cones in mice,using rAAV vectors expressing green fluorescent protein (GFP).

The constructs are packaged in a rAAV capsid and tested in vivo in amouse model. As shown in FIGS. 5 and 6, four rAAV vectors, i.e.rAAV5-CMVenh-PR1.1-GFP, rAAV5-PR1.5-GFP, rAAV5-PR1.7-GFP, andrAAV5-PR2.1-GFP, are produced by a standard plasmid transfection method.The rAAV vectors that have been packaged in transfected cells areharvested by cell lysis and then purified by iodixanol (IDX) gradientfollowed by Q Sepharose HP column chromatography, and formulated inAlcon BSS solution. Normal mice are then injected by subretinalinjection (1 μL) in both eyes (5 mice per vector). Six weeks postinjection, mice are sacrificed, eyes enucleated and retinal sectionsprepared. Slides are stained with DAPI to identify nuclei andimmunostained for GFP and for PNA (a marker for cone photoreceptors).The results are shown in FIGS. 5 and 6. GFP protein expression wasdetected in photoreceptors (cones and rods) of eyes received rAAV5-GFPvectors containing one of the four promoters, i.e. PR1.1, PR1.5, PR1.7,or PR2.1. In which, PR1.5 is a relatively weaker promoter, and PR1.1 isa strong promoter but has off target GFP expression in RPE cells.Overall, PR1.7 is comparable to the PR2.1 promoter in terms of strength(both score +++ in GFP expression level in cones) and cell typespecificity (target to cones and also rods, but not RPE cells).

Example 2: Evaluation in Non-Human Primates

Further studies were carried out to evaluate three cone-specificpromoters and three AAV capsid serotypes by comparing their efficiencyand specificity to target L, M and S cones in nonhuman primates (NHP),using rAAV vectors expressing green fluorescent protein (GFP). In thefirst study, six cynomolgus macaques received bilateral subretinalinjections of AAV2tYF-GFP containing a PR1.7, CSP, or PR2.1 promoter.Each eye received two injections of 0.1 mL of AAV vector at aconcentration of 5×10¹¹ vg/mL (two 0.05 mL blebs/eye, 1×10¹¹ vg/eye).Twelve weeks post treatment, retinal tissue was obtained forquantitative reverse transcriptase PCR (qRT-PCR) andimmunohistochemistry. The vector with the PR1.7 promoter was found toresult in robust and specific targeting of GFP-reporter gene expression(Grade 3) in all three types of cones in the subretinal bleb areas inall NHP eyes. FIG. 7 shows the results of in-life fundusautofluorescence imaging (FAF) to detect the presence of fluorophores(GFP) in the eye. Variable staining of GFP (Grades 0, 1 or 2) was seenin the subretinal bleb areas in the PR2.1 promoter group and no GFPlabeling was present in any of the eyes receiving the CSP promoter group(Grade 0) (FIG. 8, Table 1, below). Table 1 is a summary of the hereindescribed Immunohistochemistry Grading in Promoter Selection Study. InTable 1, GFP expression was graded as 0 (no staining), 1 (mildstaining), 2 (moderate staining) and 3 (intense staining).

TABLE 1 Group Eye Promoter RG1 B1 RG2 B2 Mean Mean GFP + RG GFP + BI00253 OS None na na 0 0 0 0 na na IM080058 None na na 0 0 0 na na OSIM083748 None na na 0 0 0 na na OS I08050 OS AAV2tYF-CSP-GFP 0 0 0 0 0 0no no I08051 OD AAV2tYF-CSP-GFP 0 0 0 0 0 no no I08053 ODAAV2tYF-CSP-GFP 0 0 0 0 0 no no I08054 OS AAV2tYF-CSP-GFP 0 0 0 0 0 nono I08053 OS AAV2tYF-PR2.1- 1 1 0 0 0.5 1.4 yes no GFP I08050 ODAAV2tYF-PR2.1- 1 2 1 1 1.25 yes yes GFP I08052 OD AAV2tYF-PR2.1- 2 2 1 11.5 yes no GFP I08049 OS AAV2tYF-PR2.1- 3 2 2 2 2.25 yes yes GFP I08049OD AAV2tYF-PR1.7- 3 3 3 3 3 3 yes yes GFP I08051 OS AAV2tYF-PR1.7- 3 3 33 3 yes yes GFP I08052 OS AAV2tYF-PR1.7- 3 3 3 3 3 yes yes GFP I08054 ODAAV2tYF-PR1.7- 3 3 3 3 3 yes yes GFP

Taken together, the results of these experiments show that the strengthand specificity of shortened PR1.7 is comparable to that of PR2.1 inmice. It was found that the PR1.7 promoter directed the highest level ofexpression ni reg/green and blue cones. The CNGB3 native promoter hasbeen identified to be a strong RPE-specific promoter in mice.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A nucleic acid comprising a portion of the cone cell specificpromoter PR 2.1.
 2. The nucleic acid of claim 1, comprising the sequenceSEQ ID NO:
 4. 3. The nucleic acid of claim 1, wherein PR2.1 is truncatedat the 5′ or the 3′ end.
 4. The nucleic acid of claim 3, wherein thetruncation is between about 100 base pairs to 1,500 base pairs. 5.(canceled)
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled) 10.(canceled)
 11. (canceled)
 12. (canceled)
 13. (canceled)
 14. A nucleicacid comprising a nucleotide sequence selected from a nucleotidesequence of SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO:
 3. 15. (canceled)16. (canceled)
 17. (canceled)
 18. The nucleic acid of claim 1, whereinthe promoter is capable of promoting CNGB3 expression in S-cone cells,M-cone cells, and L-cone cells.
 19. The nucleic acid of claim 1, whereinthe promoter is capable of promoting CNGA3 expression in S-cone cells,M-cone cells, and L-cone cells.
 20. The nucleic acid of claim 1, whereinthe promoter is capable of promoting GNAT2 expression in S-cone cells,M-cone cells, and L-cone cells.
 21. A recombinant adeno-associated(rAAV) expression vector comprising a target nucleic acid sequenceoperably linked to the nucleic acid of claim
 1. 22. (canceled) 23.(canceled)
 24. (canceled)
 25. (canceled)
 26. The expression vector ofclaim 21, wherein the target nucleic acid sequence encodes a cyclicnucleotide-gated channel subunit B (CNGB3) polypeptide.
 27. (canceled)28. (canceled)
 29. (canceled)
 30. The expression vector of claim 21,wherein the target nucleic acid sequence encodes a cyclicnucleotide-gated channel subunit A (CNGA3) polypeptide.
 31. (canceled)32. (canceled)
 33. (canceled)
 34. The expression vector of claim 21,wherein the target nucleic acid sequence encodes a Guaninenucleotide-binding protein G(t) subunit alpha-2 (GNAT-2) polypeptide.35. (canceled)
 36. (canceled)
 37. (canceled)
 38. A mammalian cellcomprising the expression vector of claim
 21. 39. A transgene expressioncassette comprising: (a) the nucleic acid of claim 1; (b) a nucleic acidselected from the group consisting of a CNGB3 nucleic acid, a CNGA3nucleic acid, and a GNAT2 nucleic acid; and (c) minimal regulatoryelements.
 40. A nucleic acid vector comprising the expression cassetteof claim
 39. 41. The vector of claim 40 wherein the vector is anadeno-associated viral (AAV) vector.
 42. A kit comprising the expressionvector of claim 21 and instructions for use.
 43. A method of treating aneye disease comprising administering to a subject in need thereof theexpression vector of claim 21, thereby treating the subject.
 44. Amethod of promoting CNGA3 or CNGB3 expression in the cone cells of asubject comprising administering to the subject the expression vector ofclaim 21, thereby promoting CNGA3 or CNGB3 expression.
 45. The method ofclaim 43, wherein the eye disease is associated with a genetic mutation,substitution, or deletion that affects retinal cone cells. 46.(canceled)
 47. (canceled)
 48. (canceled)
 49. (canceled)
 50. (canceled)51. (canceled)